Bacteriophages have been used in strategies for detecting molecules-of-interest. For example, a method employing the bacteriophage M13 has been used to assay for various proteins of interest. In this method M13 phage displaying peptides fused to pIII, a minor M13 coat protein, have been used to screen for protein binding molecules and antibodies (Scott et al. (1990) Science 249:386; Devlin et al. (1990) Science 249:404). Special M13-derived systems have been used to express antibodies as fusion proteins on the surface of the phage, and techniques have been developed to enrich the population for phage expressing antibodies with desired affinities for an antigen (Garrard et al. (1991) Bio/Technol 9:1373; Barbas et al. (1991) Proc. Natl. Acad. Sci (USA) 88:7978). However, the use of M13 in assay methods is limited because M13 infection is not immediately ascertainable. This is because infection by M13 does not provide the cell with compounds required for cell growth and does not lyse the cells.
The bacteriophage T4 has also been used in assays for various proteins. For example, T4 has been used to detect nerve growth factor (NGF) (Oger. et al. (1974) Proc. Natl. Acad. Sci. (USA) 71:1554-1558). In this assay, T4 was chemically coupled to NGF using glutaraldehyde. Glutaraldehyde reacts with lysine and cysteine residues as well as with .alpha.-NH.sub.3 groups (see Laboratory Techniqus Biochemistry and Molecular Biology (Elsevier Science Publishing Co., Amsterdam (1988)) Vol. 19). Antibodies against NGF were used to render the bacteriophage non-infective. When NGF was added to the medium, T4-NGF was displaced from the antibody and was free to infect Escherichia coli (E. coli).
Bacteriophage T4 has also been used to detect antibodies against a wide range of compounds. For example, Becker et al. (Immunochem. (1970) 7:741) used a T4 bacteriophage to detect antibodies against p-azobenzenearsonate. Hurwitz et al. (Eur. J. Biochem. (1970) 17:273) used a bacteriophage to detect and estimate levels of angiotensin-II-beta-amide and its antibodies. Gurari et al. (Eur. J. Biochem. (1972) 26:247) used bacteriophage T4 in the detection of antibodies to nucleic acids. These detection methods involve the chemical modification of the T4 phage resulting in the non-specific exposure on the phage surface of a compound to which the antibodies to be assayed are targeted. Such antibodies render the bacteriophage non-infective, thus enabling the decrease in plaque formation to be used as a measure of the level of antibody present.
The T4 system has also been used to measure hapten concentrations (see, e.g., Hurwitz et al. (1970) Eur. J. Biochem. 17:273-277) In this system, T4 is chemically modified such that it exposes the desired hapten non-specifically on its surface. The addition of anti-hapten antibody destroys the infectivity of the phage. Infectivity is restored in the presence of hapten.
Although both the M13 and T4 phage systems can be used to detect the presence of a compound by their ability to become infected in the presence of that compound, infection by M13 is normally not immediately ascertainable, and T4 infection is lethal. Thus, these systems cannot be used where a quick screening or selection method based on the survival of the infected bacterial cell is desired, such as where a particular cell type is being selected, or when the object of phage infection is to restore the ability of an auxotrophic bacterial cell to survive on its own under a given set of growth conditions. Another limitation is that none of these methods of detection allow the in vitro assembly of phage, and none allow the display of non-proteinaceous target molecules on a particular area of a phage. Particular M13-derived phagemid systems,such as the one employed by Barbas et al. (Proc. Natl. Acad. Sci, USA (1991) 88:7978) do carry genes which would endow an infected cell with a selective growth advantage. However, these systems are limited to the expression of peptides or proteins. Furthermore, in all M13 systems where fusion proteins have been used to display proteins, the displayed protein has always been the molecule-of-interest and therefore not useful for the detection of such molecules.
Thus, what is needed are efficient and accurate assay methods utilizing bacteriophage infection that give fast results and that do not have to result in bacterial cell death. Additionally, assay methods utilizing bacteriophage infection are needed for non-proteinaceous molecules of interest and for living cells. In addition, novel selection and screening techniques are needed for the continuous production of desirable compounds by cell lines.